Analysis on the mechanisms of NF-κB/Reactive Oxigen Species-mediated survival of hematopoietic cells and its clinical application

为了研究NF-κB家族蛋白在造血中的作用，我们首先在因子依赖性细胞系Ba/F3中以诱导方式表达显性阴性Rel/NF-κB (i -κ bsr)，它可以抑制所有NF-κB家族蛋白的功能。虽然诱导的i - κ bsr既不影响血小板生成素依赖性的生长，也不影响gp130介导的Ba/F3细胞的生长，但它在低浓度下抑制了白细胞介素-3和红细胞生成素依赖性的生长。此外，i - κ bsr增强了因子剥夺型凋亡，这与线粒体膜电位的降低和活性氧（ROS）的积累有关。由于i - κ bsr增强的细胞凋亡几乎完全被活性氧清除酶如MnSOD和硫氧还蛋白X （TRX）所抵消，因此ROS可能参与了i - κ bsr增强的细胞凋亡。当i - κ bsr在正常造血干细胞/祖细胞中表达时，即使在适当的细胞因子存在的情况下，i - κ bsr也能通过积累ROS诱导细胞凋亡，ROS清除酶、MCI-186、n -乙酰半胱氨酸（NAC）和TRX也能增强这种作用。对于IκBSR诱导ROS积累和凋亡的机制，半定量逆转录- pcr分析显示，IκBSR抑制了ROS清除酶（MnSOD、谷胱甘肽过氧化物酶、TRX）的表达。此外，抗凋亡Bcl-2家族成员（Bcl-2、Bcl-XL和A1）的表达也受到抑制。接下来，我们使用OP9系统在造血的不同阶段以诱导方式表达i - κ bsr，其中造血细胞被诱导从胚胎干细胞发育而来。当i - κ bsr在Flk-1^+细胞（推定成血管细胞）阶段表达时，i - κ bsr通过ROS积累诱导细胞凋亡，从而抑制原始造血祖细胞的发育。此外，当IκBSR在造血祖细胞发育后表达时，可通过ROS积累诱导细胞凋亡，从而抑制造血祖细胞向红细胞、巨核细胞和粒细胞的终末分化。这些结果表明，NF-κB通过消除ROS在造血过程的多个步骤中阻止细胞凋亡是必需的。少

To examine the roles for NF-κB family proteins in hematopoiesis, we first expressed dominant negative Rel/NF-κB (IκBSR), which can inhibit the function of all NF-κB family proteins, in a factor-dependent cell line, Ba/F3, in an inducible manner. Although the induced IκBSR neither affected thrombopoietin-dependent nor gp130-mediated growth of Ba/F3 cells, it suppressed interleukin-3- and erythropoietin-dependent growth at low concentrations. In addition, IκBSR enhanced factor-deprived apoptosis, which was associated with the reduction of the mitochondrial membrane potential and accumulation of reactive oxygen species (ROS). Since IκBSR-enhanced apoptosis was cancelled by ROS scavenger enzymes such as MnSOD and thioredoxin X (TRX) almost completely, ROS were supposed be involved in the IκBSR-enhanced apoptosis. When IκBSR was expressed in normal hematopoietic stem/progenitor cells, IκBSR induced apoptosis even in the presence of appropriate cytokines by accumulating ROS, which was also r … More elieved by ROS scavenger enzymes, MCI-186, N-acetyl-cysteine (NAC), and TRX. As for the mechanism of the IκBSR-induced ROS accumulation and apoptosis, semiquantitative reverse transcriptese-PCR analyses showed that the expression of ROS scavenger enzymes (MnSOD, glutathione peroxidase, and TRX) was suppressed by IκBSR. In addition, the expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL and A1) was also suppressed. Next, we expressed IκBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from embryonic stem cells. When IκBSR was expressed at the stage of Flk-1^+ cells (putative hemangioblasts), IκBSR inhibited the development of primitive hematopoietic progenitor cells by inducing apoptosis through the ROS accumulation. Furthermore, when IκBSR was expressed affer the development of hematopoietic progenitor cells, it inhibited their terminal differentiation toward erythrocytes, megakaryocytes, and granulocytes by inducing apoptosis through the ROS accumulation. These results indicate that NF-κB is required for preventing apoptosis at multiple steps of hematopoiesis by eliminating ROS. Less